1. Field of the Invention
This invention relates to a method to assess oxidation of lipids and fats ex vivo by quantification of prostaglandin-like compounds and their metabolites produced by a noncyclooxygenase free radical catalyzed mechanism.
2. Description of Related Art
Free radicals derived primarily from oxygen have been implicated in the pathophysiology of a number of human diseases, such as atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, cancer and aging. A variety of methods have been developed to assess oxidative stress and more generally, oxidation or autooxidation of lipids and fats ex vivo i.e. food spoilage; however, some of these methods have limited sensitivity or specificity, while others are either too invasive or not adaptable for human investigation. (Halliwell, et al, 1987)
Free radicals are generally short lived and thus, indirect methods of detection are required (see Pryor, W., 1989) Standard detection methods include: electron spin resonance (directly), electron spin resonance (spin trapping), thiobarbituric acid reactive substances (TBARS), detection of malonaldehyde by direct methods (such as HPLC of malonaldehyde itself or as its dinitrophenylhydrazone), detection of other oxidation products from polyunsaturated fatty acids (such as 4-hydroxynonenal), measurement of lipid hydroperoxides, detection of volatile hydrocarbons (ethane, pentane and ethylene), detection of oxidation products from lipids other than polyunsaturated fatty acids (e.g., cholesterol), oxidation of methional, methionine, or 2-keto-4-thiomethylbutanoic acid to ethylene, oxidation of benzoic acid to carbon dioxide (often with radiolabelled carbon dioxide), oxidation of phenol, benzoic acid, or aspirin to hydroxylated products, determination of decreases in antioxidant levels (e.g., decreased GSH, tocopherol, or ascorbate) or of increases in the oxidized products from antioxidants (e.g., tocopherol quinone or the ascorbyl radical), detection of oxidized DNA bases (e.g., thymine glycol, 8-hydroxydeoxyguanosine), detection of oxidized products from proteins (e.g., methionine sulfoxide from methionine) or of proteins oxidized to carbonyl-containing products that then react with hydride-reducing agents, detection of adducts of DNA bases (e.g., by enzymatic hydrolysis post-labeling using P32), and chemiluminescence methods. (Pryor, 1989).
Unfortunately, oxidative stress is difficult to assess in humans due to lack of reliable methods to assess oxidant stress in vivo. There is also no marketed assessment for food spoilage. As one author stated, "one of the greatest needs in the field now is the availability of non-invasive test to probe the oxidative stress status of humans." (Pryor, 1989)
Morrow, et al. (1990) discovered that a series of prostaglandin F.sub.2 -isprostanes, were generated in human plasma during storage at -20.degree. C. for several months or in plasma that had been repeatedly frozen and thawed. Morrow et al. (1990) determined that these compounds were formed by a non-cyclooxygenase mechanism by autooxidation of arachidonic acid contained in plasma. This article demonstrated that prostaglandins could be generated by autooxidation during storage of biological samples which could result in artifactual results with measurements of prostaglandins in stored samples. At that time, there was nothing to suggest that this was anything more than just a non-enzymatic in vitro artifact or phenomenon that occurred during the storage of plasma or other lipid containing biological fluids. In fact, this process, autooxidation of lipids or fats, is a major process responsible for spoilage of food during storage.